Academic Background

Conjunctival Melanoma (COM) is a rare but potentially lethal ocular cancer, particularly when metastasis occurs, with limited treatment options. Although current treatments for primary conjunctival melanoma are relatively effective, once metastasis occurs, patient survival rates drop significantly, with most patients surviving less than 10 years. Therefore, a deeper understanding of how conjunctival melanoma spreads from its primary site to form metastases is crucial for developing new therapeutic strategies.

Previous studies have shown that primary conjunctival melanoma expresses Vascular Endothelial Growth Factor (VEGF) and may recruit pro-tumor M2-type macrophages. However, due to the lack of suitable models, the specific role of angiogenesis in the metastatic dissemination of conjunctival melanoma remains unclear. Angiogenesis is a key process in tumor growth, dissemination, and metastasis, often considered a precursor to tumor metastasis. Most solid tumors require a vascular system to supply sufficient oxygen, nutrients, and waste disposal, making angiogenesis essential for sustaining tumor cell proliferation.

Additionally, immune cells in the tumor microenvironment, particularly macrophages, are believed to be important regulators of angiogenesis. Macrophages in tumors are known as Tumor-Associated Macrophages (TAMs) and are typically categorized into two subtypes: M1 and M2. M1 macrophages have anti-tumor or pro-inflammatory effects, while M2 macrophages are considered pro-tumor or anti-inflammatory. M2 macrophages participate in tumor angiogenesis by secreting pro-angiogenic factors such as VEGF and TGF-β.

Although M2 macrophages have been linked to increased tumor angiogenesis in uveal and cutaneous melanomas, their role in conjunctival melanoma remains controversial. Some studies suggest no correlation between the presence of macrophages and conjunctival melanoma prognosis, while others report an association between the absence of inflammatory cells and poor prognosis.

Source of the Paper

This paper was co-authored by Jie Yin, Gabriel Forn-Cuní, Akshaya Mahalakshmi Surendran, Bruno Lopes-Bastos, Niki Pouliopoulou, Martine J. Jager, Sylvia E. Le Dévédec, Quanchi Chen, and B. Ewa Snaar-Jagalska from Leiden University in the Netherlands. The paper was published online on June 6, 2024, in the journal Angiogenesis, with the DOI 10.1007/s10456-024-09930-y.

Research Process and Results

Research Process

1. Cell Culture and Treatment
   The study used two conjunctival melanoma cell lines (CRMM1 and CRMM2) and two breast cancer cell lines (4T1 and 67NR). These cells were cultured in specific media and labeled with far-red fluorescence via lentiviral transduction for tracking in the zebrafish model.

2. Zebrafish Model and Transplantation
   The study used transgenic zebrafish embryos with fluorescently labeled blood vessels and macrophages. Conjunctival melanoma cells were injected into the perivitelline space (PVS) of the zebrafish embryos, and the dynamic processes of angiogenesis and macrophage recruitment were observed using confocal microscopy.

3. Macrophage Ablation and Glycolysis Inhibition
   To study the role of macrophages in angiogenesis, the researchers used the NSFB nitroreductase (NTR)/metronidazole (MTZ) system to ablate macrophages in zebrafish embryos. Additionally, glycolysis was inhibited using 2-deoxy-D-glucose (2DG) and GSK2837808A to observe its effects on angiogenesis.

4. Lactate Assay and Macrophage Polarization
   Lactate levels in cell culture supernatants were measured using a lactate assay kit, and the expression of macrophage polarization markers and pro-angiogenic factors was analyzed via qPCR.

Key Results

1. Conjunctival Melanoma Cells Induce Angiogenesis and Macrophage Recruitment
   The study found that conjunctival melanoma cells induced a strong angiogenic response in the zebrafish model and recruited a large number of macrophages. Through time-lapse imaging, researchers observed that macrophages rapidly aggregated at the site of injected cells within 2 hours and co-localized with cancer cells within 24 hours.

2. Angiogenic Response is Macrophage-Dependent
   By chemically ablating macrophages, the study found that the angiogenic response induced by conjunctival melanoma cells was significantly reduced. This indicates that macrophages play a crucial role in the angiogenesis induced by conjunctival melanoma cells.

3. Lactate-Driven Macrophage Polarization and Angiogenesis
   The study found that conjunctival melanoma cells produce lactate through glycolysis, which recruits and polarizes macrophages toward the M2 phenotype. These M2 macrophages secrete pro-angiogenic factors such as VEGF, TGF-β, and IL-10 in the tumor microenvironment, thereby inducing angiogenesis.

4. Glycolysis Inhibition Attenuates Angiogenesis
   By inhibiting glycolysis, the study found that lactate secretion by conjunctival melanoma cells was significantly reduced, and macrophage recruitment and polarization were also inhibited, ultimately leading to a weakened angiogenic response.

Conclusions and Significance

This study reveals the mechanism by which conjunctival melanoma cells produce lactate through glycolysis, recruit and polarize macrophages, and subsequently drive angiogenesis. This discovery provides new insights into the metastatic dissemination of conjunctival melanoma and offers a theoretical foundation for developing therapeutic strategies targeting glycolysis and macrophage polarization.

Research Highlights

1. Novel Zebrafish Model: The study used a transgenic zebrafish model with fluorescently labeled blood vessels and macrophages to observe the dynamic interactions between tumor cells, macrophages, and angiogenesis in real-time.
2. Lactate-Driven Macrophage Polarization: For the first time in a zebrafish model, the study confirmed lactate as a key signaling molecule for macrophage recruitment and polarization, revealing its important role in tumor angiogenesis.
3. Potential Therapeutic Value of Glycolysis Inhibition: The study showed that inhibiting glycolysis significantly attenuates angiogenesis induced by conjunctival melanoma cells, providing experimental evidence for developing new therapeutic strategies.

Other Valuable Information

The study also found that conjunctival melanoma cells maintained high glycolytic properties in the zebrafish xenograft model, with lactate secretion levels comparable to the highly metastatic breast cancer cell line 4T1. This finding further supports the important role of lactate in the tumor microenvironment.

Summary

This study, using a zebrafish model, delves into the mechanism by which conjunctival melanoma cells produce lactate through glycolysis, recruit and polarize macrophages, and subsequently drive angiogenesis. This discovery not only enhances our understanding of the metastatic dissemination of conjunctival melanoma but also provides important experimental evidence for developing new therapeutic strategies.